Journal: Biochimica et biophysica acta

Article Title: Targeted delivery of indinavir to HIV-1 primary reservoirs with immunoliposomes.

doi: 10.1016/s0005-2736(01)00432-1

Figure Lengend Snippet: Fig. 2. In vitro e¤cacy of free indinavir (a), indinavir incorporated in liposomes (R) or anti-HLA-DR immunoliposomes (b) to in- hibit HIV-1 replication in PM1 cells. The drug concentration of indinavir in all formulations was 10 nM (A), 25 nM (B), 50 nM (C) and 100 nM (D). Uninfected cells (E) and untreated infected cells (O) were used as controls. Results represent the average of tripli- cate incubations from one experiment typical of three.

Article Snippet: Sup-T1, a human CD4 T lymphocyte cell line, PM1, a clonal derivative of HUT 78 cells, and RAJI, an Epstein^Barr virus-carrying B cell line BBAMEM 78193 14-12-01 that express high levels of cell surface HLA-DR, -DP and -DQ proteins were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

Techniques: In Vitro, Liposomes, Concentration Assay, Infection

Journal: EMBO Reports

Article Title: Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways

doi: 10.1038/embor.2010.214

Figure Lengend Snippet: Drosophila and human PMI are mitochondrial proteins. (A) Salivary gland cells from amino-terminal CFP::PMI larvae stained for ATP synthase (red). CFP::PMI (green) is targeted to mitochondria. (B) In adult myocytes, CFP::PMI (green) colocalizes with mitochondrial ATP synthase (red) and accumulates as dots. (C) actin-GAL4 UAS-PMI::mCherry, mito::GFP larval neuron. PMI::mCherry accumulated as dots along or at the tip of mitochondrial tubules (arrows). (D) C2C12 cells transfected with CFP::TMEM11 (blue) and stained with mitotracker (red) and 4,6-diamidino-2-phenylindole (DAPI; nucleus). CFP signal is shown as dots scattered throughout the cytoplasm. Bottom, CFP::TMEM11 (blue) colocalized with mitochondria (red, arrows), and often accumulated as dots along mitochondrial tubules (small arrowheads).

Article Snippet: For western blot, we used TMEM11 antibody (1/500; Proteintech). siRNA for luciferase and TMEM11 (si1: J-00540-09 and si2: J-005440-10) were from Dharmacon Research.

Techniques: Staining, Transfection

Journal: EMBO Reports

Article Title: Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways

doi: 10.1038/embor.2010.214

Figure Lengend Snippet: Drosophila and human PMI localized at the mitochondrial inner membrane. (A) Transmission electron microscopy (TEM) images of CFP::PMI adult flight muscle labelled with GFP (CFP::PMI), ATP synthase or VDAC antibodies. Secondary antibody is conjugated to 10 (CFP::PMI and VDAC) or 15 nm (ATP synthase) gold beads. Similarly to inner-membrane protein ATP synthase, CFP::PMI staining is inside the mitochondria (arrows) in areas in which cristae are visible (higher magnification). The outer-membrane protein VDAC is detected all around the mitochondria (arrows). (B) Gold bead distribution for CFP::PMI (amino-terminal), PMI::CFP (carboxy-terminal), ATP synthase and VDAC immunolabelling. Mitochondria analysed: CFP-tagged PMI (n=59), ATP synthase (n=28), VDAC (n=28). Beads counted: CFP-tagged PMI (n=736), ATP synthase (n=900), VDAC (n=1034). Student's t-test: **** P<0.0001; three independent stainings. (C) Identical cell-equivalent amounts of HeLa cells post-nuclear supernatant (TOT, 100 μg of protein) separated into cytosol plus light membranes (CYT) and mitochondrial (MIT) fractions were analysed by western blot using TMEM11, mitochondrial HSP60 and actin antibodies. Results demonstrate that TMEM11 is a mitochondrial protein. (D) Mitochondria were incubated in the absence or presence of trypsin and triton. Samples were analysed by western blot using HSP60 (matrix), VDAC (outer membrane), Smac/DIABLO (intermembrane space), COX4 (inner membrane) and TMEM11 antibodies. Results demonstrate that TMEM11 is localized inside the mitochondria. (E) Mitochondria incubated or not in hypo-osmotic buffer (osmotic shock) were incubated in the absence or presence of trypsin, triton and increasing concentrations of Digitonin. Samples were analysed by western blot using HSP60 (matrix), DRP1 (outer membrane), Smac/DIABLO (intermembrane space) and TMEM11 antibodies. These results show that TMEM11 is a matricial protein. (F) Mitochondria (intact or sonicated) were incubated in hypo-osmotic buffer (MB/10) alone or supplemented by 1-M NaCl or 0.1-M Na2CO3, pH 11, and centrifuged to separate membrane pellets (P) from soluble protein supernatants (S). Samples were analysed by western blot using VDAC, TMEM11 and cytochrome C antibodies. With both treatments, TMEM11 remained in membrane pellets as did the integral membrane protein VDAC, whereas cytochrome C, peripherally associated to the inner membrane, was readily extracted by either treatment. This demonstrates that TMEM11 is a transmembrane protein.

Article Snippet: For western blot, we used TMEM11 antibody (1/500; Proteintech). siRNA for luciferase and TMEM11 (si1: J-00540-09 and si2: J-005440-10) were from Dharmacon Research.

Techniques: Membrane, Transmission Assay, Electron Microscopy, Staining, Western Blot, Incubation, Sonication

Journal: EMBO Reports

Article Title: Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways

doi: 10.1038/embor.2010.214

Figure Lengend Snippet: TMEM11 is required to shape mitochondria in human cells. (A) Human cells co-transfected with either luciferase siRNA or siRNA targeting TMEM11, dynamin-related protein 1 (DRP1) or OPA1 and a mito::DsRed reporter construct. In control cells (luc_si), mitochondria are organized into a dense network of tubules. In cells treated with TMEM11_siRNA (two different siRNAs tested), mitochondria condense into large spherical entities that aggregate around the nucleus. This is referred to as ‘balloon phenotype'. Cells treated with DRP1_siRNA have dense, spherical mitochondria and hyperfilamented mitochondrial tubules. Mitochondria in OPA1_siRNA-treated cells were more numerous and smaller. (B) Time-course western blot experiments showing the extinction efficiency of two siRNAs (si1, si2) on TMEM11 protein level, compared with control siRNA against luciferase (luc_si). (C) Western blot experiments showing protein levels of OPA1 and DRP1 in cells treated with specific siRNA (DRP1_si or OPA1_si) or a non-relevant siRNA (luc_si). (D) Data show quantification of mitochondrial balloon phenotype 24, 48 or 72 h after transfection. n=300 cells in each condition. (E) Mean of three independent experiments showing the percentage of cells that exhibit a balloon phenotype at 48 h when treated with TMEM11 siRNA (si1, si2) or luciferase siRNA. Differences as compared with luc_si were statistically significant: *P<0.05, **P<0.01. n=900 cells in each condition. siRNA, short-interfering RNA.

Article Snippet: For western blot, we used TMEM11 antibody (1/500; Proteintech). siRNA for luciferase and TMEM11 (si1: J-00540-09 and si2: J-005440-10) were from Dharmacon Research.

Techniques: Transfection, Luciferase, Construct, Control, Western Blot, Small Interfering RNA